Measurement of pre‐treatment inflammatory cytokine levels is valuable for prediction of treatment efficacy to tumor necrosis factor inhibitor in axial spondyloarthritis patients

Abstract Aim To evaluate the correlation of inflammatory cytokines with the treatment response to tumor necrosis factor inhibitor (TNFi) in axial spondyloarthritis (axSpA) patients. Methods This study enrolled 86 axSpA patients and 20 healthy controls (HCs). Inflammatory cytokines including tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1β, IL‐6, IL‐12, IL‐17A, IL‐21, IL‐23, and IL‐32 were determined in serum samples of axSpA patients before treatment and in HCs after enrollment. All patients received 40 mg adalimumab every 2 weeks for 12 weeks; meanwhile, ASAS40 (40 criteria of the Assessment by the SpondyloArthritis International Society) response rates were evaluated at weeks 2, 4, 8, and 12. Results Most inflammatory cytokines were elevated in axSpA patients compared with HCs (all P < 0.05) except for IL‐32 (P = 0.101). In axSpA patients, ASAS40 response rates were 0%, 19.5%, 34.5%, 47.1%, and 56.3% at weeks 0, 2, 4, 8, and 12, respectively. Baseline [interquartile range] IL‐6 (47.3 [32.5‐53.4] pg/mL vs 31.7 [23.0‐50.9] pg/mL, P = 0.005) and IL‐17A (127.9 [90.7‐149.5] pg/mL vs 96.6 [56.1‐112.6] pg/mL, P < 0.001) were higher in axSpA patients with ASAS40 response compared with those without ASAS40 response, while baseline TNF‐α, IL‐1β, IL‐12, IL‐21, IL‐23, and IL‐32 were not different between them (all P > 0.050). Multivariate logistic regression analysis disclosed that baseline IL‐17A (P = 0.037), C‐reactive protein (P = 0.012), and history of TNF inhibitor (P = 0.029) were independently associated with ASAS40 response. Furthermore, baseline IL‐17A, C‐reactive protein, history of TNFi, and their combination had an acceptable to good ability for predicting ASAS40 response. Conclusion Measurement of pre‐treatment inflammatory cytokine levels is valuable for predicting treatment efficacy of TNFi in axSpA patients.


| INTRODUC TI ON
Axial spondyloarthritis (axSpA), a chronic inflammatory disease leading to vertebral and sacroiliac fusion, is characterized by lower back pain and back stiffness in the morning. [1][2][3] Even though there is still no cure for axSpA patients, the application of tumor necrosis factorα (TNFα) inhibitor in the treatment of axSpA ameliorates the disease status, including controlling the inflammation, decreasing the disease activity, and improving the health-related quality of life. [4][5][6] However, the annual cost of TNF inhibitor is high; in addition, a certain proportion of axSpA patients stop the TNF inhibitor therapy because of the lack of efficacy and the presence of adverse events. [7][8][9] Hence, it is essential to find biomarkers to predict the TNF inhibitor response in axSpA patients.
Inflammatory cytokines, including TNFα, interleukin (IL)-1β, IL-6, IL-12, IL-17A, IL-21, IL-23, and IL-32, are involved in regulating the immunoreaction. [10][11][12][13] For instance, IL-1β may induce the inflammatory response and oxidative stress through the Rhokinase/IκBα/NF-κB activation mechanism, 10 and the activation of the IL-23/IL-17 axis may contribute to the inflammatory response by secretion of a range of pro-inflammatory cytokines (including IL-6, TNFα, and IL-1). T-cell-attracting and neutrophil-attracting chemokines including CCL2, CCL7, CXCL1, and CXCL2. 11 Previous studies have reported that IL-1β, IL-17A, and TNFα may predict the treatment response to TNF inhibitors in inflammatory or rheumatic diseases, such as psoriasis and Crohn disease. 14,15 However, their relation to the TNF inhibitor response in axSpA patients is still obscure. Therefore, this study analyzed inflammatory cytokine levels in axSpA patients treated with TNF inhibitor and further aimed to investigate their correlation with TNF inhibitor treatment response in axSpA patients.

| Participants
After being approved by the Institutional Review Board, this study consecutively recruited 86 individuals with axSpA to receive adali- (c) patients who were highly unable to benefit from adalimumab therapy (including patients who were known to have no response to previous adalimumab treatment or patients who were known to have no response to more than two kinds of other TNF inhibitors); (d) pregnant and lactating female patients. At the same time, another 20 age-(20-40 years) and gender-(male to female as 4:1 ratio) matched healthy controls (HCs) were also enrolled. The exclusion criteria were also suitable for the HCs. Each participant signed informed consent.

| Determination of inflammatory cytokines
A venous blood sample for study use was collected in a serum separator tube. Serum was isolated by centrifugal separation at 1000 g for 20 minutes, immediately used to determine inflammatory cytokines, including TNFα, IL-1β, IL-6, IL-12, IL-17A, IL-21, IL-23, and IL-32. The determination of inflammatory cytokines was completed by enzyme-linked immunosorbent assay (ELISA) using Human Quantikine ELISA Kits (R&D Systems, Minneapolis, MN, USA). The assay was carried out referring to the complete assay procedure of the kits. In brief, 100 μL of assay diluent and 50 μL of standards, control, or sample were added to each well and incubated for 2 hours. After that, each well was washed four times, and 100 μL of the conjugate was added to each well, followed by incubation for 1 hour and cleaning four times. Subsequently, 200 μL of substrate solution was added to each well, followed by incubation at room temperature for 30 minutes avoiding light.
Finally, 50 μL of stop solution was added to each well, followed by immediately reading absorbance at 450 nm. The standard curve was fitted, which was used for calculating the concentration of unknown samples.

| Administration of adalimumab and evaluation
AxSpA patients in this study received a subcutaneous injection of 40 mg adalimumab (Abbott Laboratories, Chicago, IL, USA) every 2 weeks for 12 weeks. Evaluation of treatment response was started at week 2 after initiation of adalimumab treatment, which was then performed at weeks 4, 8, and 12, respectively.
The Assessment by SpondyloArthritis International Society 40 improvement criteria (ASAS40) were adopted to evaluate treatment response. ASAS40 response was defined as the improvement of at least 40% and at least 2 units in at least three domains on a scale of 10, without worsening at all in the remaining domain, where the territories comprised global patient assessment, pain, function, and inflammation (mean of BASDAI questions 5 and 6). 16 In the analysis, patients who achieved an ASAS40 response at week 12 were classified as response patients, and the others without achievement of an ASAS40 response at week 12 were classified as non-response patients.

| Statistical analysis
The statistical analysis process and graph plotting were completed using SPSS 24.0 (IBM Corp., Armonk, NY, USA) and Graphpad prism for ASAS40 response at week 12 was performed. The forward stepwise methodology was applied to screen the independent prediction factors, further estimated for performance by receiver operating characteristic (ROC) curve analysis. The optimal specificity and sensitivity in the ROC curve analysis were calculated at the best cut-off point, which was screened out from the values that gave the maximum specificity and sensitivity. A P value less than 0.05 indicated a statistical significance.

Baseline characteristics of axSpA patients
The mean age of axSpA patients was 33.1 ± 8.  Table 1.

ASAS40 response rate to adalimumab in axSpA patients
Patients were continually followed up for a full 12 weeks and ASAS40 response rates were evaluated at several time points, which disclosed  Figure 1).

Correlation of baseline inflammatory cytokine levels with ASAS40 response in axSpA patients
Baseline  Figure S2).

Independent predictive factors for ASAS40 response in axSpA patients
As listed in Table 2 To further evaluate the independent predictive factors for ASAS40 response, a forward stepwise multivariate logistic regression analysis was conducted, which disclosed that baseline IL-17A (OR 1.013, P = 0.037) was independently associated with a higher possibility to achieve ASAS40 response in axSpA patients. As to other variables in the multivariate logistic regression model, the history of TNF inhibitor (OR 0.279, P = 0.029) was an independent factor in predicting a lower likelihood of achieving ASAS40 response in axSpA patients. In contrast, baseline CRP (OR 1.042, P = 0.012) was independently associated with a higher possibility to achieve ASAS40 response in axSpA patients ( Table 2).
The ROC curve analysis showed that baseline IL-17A, history of TNF inhibitor, baseline CRP, and their combination had an acceptable ability for predicting ASAS40 response of axSpA patients with areas under the curve of 0.740, 0.621, 0.824, and 0.847, respectively ( Figure 3). Besides, this study also determined the ability of baseline IL-6 for the prediction of ASAS40 response, which showed that its area under the curve was only 0.675 ( Figure S3), which was numerically lower than the baseline IL-17A.

| DISCUSS ION
Our study disclosed that more than 50% of axSpA patients achieved ASAS40 response at week 12 after adalimumab treatment. This discovery is similar to previous reports revealing that approximately 44.5%-53% of patients realized ASAS40 response after 12 weeks of adalimumab treatment. 17,18 Furthermore, this finding may explain that adalimumab restrains the production of inflammatory cytokines by targeting TNFα, thereby further suppressing inflammation, reducing disease activity, and improving spine function in axSpA patients. Hence, axSpA patients respond well to adalimumab.
As for the correlation between the baseline levels of inflammatory cytokines and ASAS40 response, a recent study showed that after treatment with TNF inhibitors, axSpA patients with good response at week 12 have a lower baseline IL-6 level. In contrast, the non-response patients have a higher baseline IL-6 level. 19 In our Previous studies have evaluated the value of cytokine levels in predicting the response of axSpA patients to TNF inhibitors. 19,[21][22][23][24] For instance, one study showed that CRP and Jun N-terminal kinase pathway-associated phosphatase (JKAP) might predict the treatment response to TNF inhibitors. 21 Another study exhibited that circulating baseline IL-6 and erythrocyte sedimentation rate might reflect the response to adalimumab in AS patients. 19 Partially in line with previous studies, our study found that high baseline levels of IL-17A and CRP were independently correlated with a higher ASAS40 response rate. In comparison, the history of TNF inhibitor independently predicted a lower ASAS40 response rate. Besides, the ROC curve analysis showed that baseline IL-17A, history of TNF inhibitor, baseline CRP, and their combination had acceptable excellent ability in predicting ASAS40 response. Their combination showed the most substantial ability in distinguishing patients with ASAS40 response from those without ASAS40 response. These results could be explained by the following. (a) IL-17A plays a vital role in the development of inflammation in axSpA patients; meanwhile, a high baseline IL-17A level indicates a more serious inflammatory condition, which intensifies the efficacy of the TNF inhibitors, thereby causing a higher ASAS40 response rate. 20 The ASAS40 response was evaluated after short-term treatment (12 weeks), and the value of inflammatory cytokine levels for predicting long-term treatment response to TNF inhibitor as well as the disease flare is not clear, which needed further evaluation. The inflammatory cytokine levels were only evaluated at baseline, but not during the follow-up period, so the value of inflammatory cytokine levels in monitoring the progression of axSpA needs further exploration.
Measurement of pre-treatment inflammatory cytokine levels is valuable for predicting treatment efficacy of TNF inhibitors in axSpA patients.

AUTH O R CO NTR I B UTI O N S
YT designed the study; FP, FC, and YT performed the study and analyzed the data; FP, HW, and JB drafted the paper. All authors read and approved the paper.

CO N FLI C T O F I NTE R E S T
All authors declare no conflicts of interest.

E TH I C A L A PPROVA L
This study was approved by Institutional Review Board, and each participant signed an informed consent.